Protein export across membranes is a critical process for life. In prokaryotes, multiple protein transport systems were evolved to facilitate the transfer of amphipathic and hydrophilic proteins of impressive sizes across lipid hydrophobic barriers. The simplest of these systems consist of an ATP binding Cassette (ABC) transporter which harnesses the energy of ATP hydrolysis to power the movement of cargo peptides or proteins across the cell membranes. In gram positive bacteria, ABC transporters export antimicrobial or quorum sensing peptides which serve to endow the organism with survival advantages under conditions of limited resources. The long term goal of this application is to illuminate the conformational dynamics of peptidase-containing ABC transporters (PCATs) which utilizes a built in cysteine protease domain to cleave the signal sequence of the cargo peptide prior to export. The premise of our strategy is grounded in 1) recent high resolution structures of PCATs that define their molecular architecture and set the stage for detailed investigation of the mechanism by which these transporters enable protein translocation and 2) the track record of the PI in the application of state of the art electron paramagnetic resonance (EPR) spectroscopy in conjunction with mutagenic analysis to active transporters. The specific aims are designed to address unanswered questions in the field including the structural basis of alternating access powered by ATP turnover, the determinants of cargo protein specificity, and the structure and environment of the cargo protein as it transitions through the transporter. Over the last decade, our approach integrated with molecular modeling has been instrumental in revealing the structural dynamics of multidrug ABC exporters and providing insight into the mechanistic diversity within this family. The significance of the proposed research stems from a molecular target at the junction of antibacterial peptide transport, bacterial immunity and represents a fundamental biochemical process by which proteins are exported across lipid bilayers.